This invention relates to a method of introducing a sample into the Micro Liquid Chromatography (hereinafter simply called MLC) and an apparatus therefor, more particularly to a method and an apparatus therefor of introducing extremely small amount of the sample accurately and in good reproductivity in a MLC, wherein a micro amount of sample less than 1 .mu.l is introduced for developing the same, by means of employing syringe driven by a pulse-motor, i.e., a stepping motor for the supplying of the mobile phase, causing the syringe by inverse rotation of the pulse-motor which has been given a predetermined number of pulses, and introducing predetermined amount of the sample into the chromatographic system.
In MLC columns less than 1 mm in inside diameter, less than 30 cm in length, for example 10-15 cm, are generally used; and micro analyses of samples less than 1 .mu.l, particularly in the range between 0.01 and 0.3 .mu.l, are usual. Since the flow rate is also so minute as 2-16 .mu.l/min, MLC is generally extremely micro or minute at the present time, and even called the High Speed Micro Liquid Chromatography.
So the conventional ways of sample picking-up and sample introduction in a liquid chromatography, which have been depending upon a micro syringe used for a large-sized liquid chromatography wherein, for example, the inside diameter of the column is in the range of 2-3 mm, the length of the column is in the range of 25-100 cm, and the flow rate is in the range of 0.5-2 ml/min, are not necessarily practicable for the present day liquid chromatography. Those traditional ways of sample introduction, when applied to the abovementioned present day MLC, have many disadvantages as follows:
(1) Since the micro syringe determines the sample amount by the graduation inpressed thereon, it is quite difficult to exactly pick up a micro amount of the sample less than 1 .mu.l and to introduce it into the chromatographic system. Personal variation in the handling amount is also inevitable in this case.
(2) The thickness of a syringe needle in the micro syringe is innegligibly great against the inside diameter of the column less than 1 mm, for example, of 0.5 mm; so the liquid is liable to be so disturbed while the sample is introduced and the needle is removed, as to affect the chromatogram obtained from the development of the sample.
(3) When a micro column of small inside diameter is used for introducing micro amount of the sample, it is required to minimize the dead volume above and below the column to the greatest extent for preventing the diffusion of the sample. In the introduction of the sample by a conventional micro syringe, an injection port is needed to be disposed at the inlet of the column, so it is extremely difficult again to reduce the dead volume here.